Search

Background: Glucagon serves as an important regulatory hormone for regulating blood glucose concentration with tight feedback control exerted by insulin and glucose. There are critical gaps in our understanding of glucagon kinetics, pancreatic α cell function and intra-islet feedback network that are disrupted in type 1 diabetes. This is important for translational research applications of evolving dual-hormone (insulin + glucagon) closed-loop artificial pancreas algorithms and their usage in type 1 diabetes. Thus, it is important to accurately measure glucagon kinetics in vivo and to develop robust models of glucose-insulin-glucagon interplay that could inform next generation of artificial pancreas algorithms. Methods: Here, we describe the administration of novel 13C15N heavy isotope-containing glucagon tracers—FF glucagon [(Phe 6 13C9,15N; Phe 22 13C9,15N)] and FFLA glucagon [(Phe 6 13C9,15N; Phe 22 13C9,15N; Leu 14 13C6,15N; Ala 19 13C3)] followed by anti-glucagon antibody-based enrichment and LC–MS/MS based-targeted assays using high-resolution mass spectrometry to determine levels of infused glucagon in plasma samples. The optimized assay results were applied for measurement of glucagon turnover in subjects with and without type 1 diabetes infused with isotopically labeled glucagon tracers. Results: The limit of quantitation was found to be 1.56 pg/ml using stable isotope-labeled glucagon as an internal standard. Intra and inter-assay variability was < 6% and < 16%, respectively, for FF glucagon while it was < 5% and < 23%, respectively, for FFLA glucagon. Further, we carried out a novel isotope dilution technique using glucagon tracers for studying glucagon kinetics in type 1 diabetes. Conclusions: The methods described in this study for simultaneous detection and quantitation of glucagon tracers have clinical utility for investigating glucagon kinetics in vivo in humans. [ABSTRACT FROM AUTHOR]

The phosphaturic activity of intact, full-length, fibroblast growth factor-23 (FGF-23) is well documented. FGF-23 circulates as the intact protein and as fragments generated as the result of proteolysis of the full-length protein. To assess whether short fragments of FGF-23 are phosphaturic, we compared the effect of acute, equimolar infusions of full-length FGF-23 and various FGF-23 fragments carboxyl-terminal to amino acid 176. In rats, intravenous infusions of full-length FGF-23 and FGF-23 176–251 significantly and equivalently increased fractional phosphate excretion (FE Pi) from 14 ± 3 to 32 ± 5% and 15 ± 2 to 33 ± 2% ( p < 0.001), respectively. Chronic administration of FGF-23 176–251 reduced serum Pi and serum concentrations of 1α,25-dihydroxyvitamin D. Shorter forms of FGF-23 (FGF-23 180–251 and FGF-23 184–251) retained phosphaturic activity. Further shortening of the FGF-23 carboxyl-terminal domain, however, abolished phosphaturic activity, as infusion of FGF-23 206–251 did not increase urinary phosphate excretion. Infusion of a short fragment of the FGF-23 molecule, FGF-23 180–205, significantly increased FE Pi in rats and reduced serum Pi in hyperphosphatemic Fgf-23 −/− knockout mice. The activity of FGF-23 180–251 was confirmed in opossum kidney cells in which the peptide reduced Na+-dependent Pi uptake and enhanced internalization of the Na+-Pi IIa co-transporter. We conclude that carboxyl terminal fragments of FGF-23 are phosphaturic and that a short, 26-amino acid fragment of FGF-23 retains significant phosphaturic activity. [ABSTRACT FROM AUTHOR]

Macropinocytosis supports the metabolic requirement of RAS-transformed pancreatic ductal adenocarcinoma cells (PDACs). However, regulators of RAS-transformation (activation) that lead to macropinocytosis have not been identified. Herein, we report that UBAP2 (ubiquitin-binding associated protein 2), regulates the activation of KRAS and macropinocytosis in pancreatic cancer. We demonstrate that UBAP2 is highly expressed in both pancreatic cancer cell lines and tumor tissues of PDAC patients. The expression of UBAP2 is associated with poor overall survival in several cancers, including PDAC. Silencing UBAP2 decreases the levels of activated KRAS, and inhibits macropinocytosis, and tumor growth in vivo. Using a UBAP2-deletion construct, we demonstrate that the UBA-domain of UBAP2 is critical for the regulation of macropinocytosis and maintaining the levels of activated KRAS. In addition, UBAP2 regulates RAS downstream signaling and helps maintain RAS in the GTP-bound form. However, the exact mechanism by which UBAP2 regulates KRAS activation is unknown and needs further investigation. Thus, UBAP2 may be exploited as a potential therapeutic target to inhibit macropinocytosis and tumor growth in activated KRAS-driven cancers. [ABSTRACT FROM AUTHOR]

Urinary exosome-like vesicles (ELVs) are a heterogenous mixture (diameter 40-200 nm) containing vesicles shed from all segments of the nephron including glomerular podocytes. Contamination with Tamm-Horsfall protein (THP) oligomers has hampered their isolation and proteomic analysis. Here we improved ELV isolation protocols employing density centrifugation to remove THP and albumin, and isolated a glomerular membranous vesicle (GMV)-enriched subfraction from 7 individuals identifying 1830 proteins and in 3 patients with glomerular disease identifying 5657 unique proteins. The GMV fraction was composed of podocin/podocalyxin-positive irregularly shaped membranous vesicles and podocin/podocalyxin-negative classical exosomes. Ingenuity pathway analysis identified integrin, actin cytoskeleton, and Rho GDI signaling in the top three canonical represented signaling pathways and 19 other proteins associated with inherited glomerular diseases. The GMVs are of podocyte origin and the density gradient technique allowed isolation in a reproducible manner. We show many nephrotic syndrome proteins, proteases, and complement proteins involved in glomerular disease are in GMVs and some were only shed in the disease state (nephrin, TRPC6, INF2 and phospholipase A2 receptor). We calculated sample sizes required to identify new glomerular disease biomarkers, expand the ELV proteome, and provide a reference proteome in a database that may prove useful in the search for biomarkers of glomerular disease. [ABSTRACT FROM AUTHOR]

Abstract: Thyroglobulin (Tg), a homodimer of 660 kD comprising 2748 amino acids, is the largest autoantigen known. The prevalence of autoimmune thyroid disease, including Hashimoto’s thyroiditis and Graves’ disease, has provided the impetus for identifying pathogenic T cell epitopes from human Tg over two decades. With no known dominant epitopes, the search has long been a challenge for investigators. After identifying HLA-DRB1∗03:01 (HLA-DR3) and H2Eb as susceptibility alleles for Tg-induced experimental autoimmune thyroiditis in transgenic mouse strains, we searched for naturally processed T cell epitopes with MHC class II-binding motif anchors and tested the selected peptides for pathogenicity in these mice. The thyroiditogenicity of one peptide, hTg2079, was confirmed in DR3 transgenic mice and corroborated in clinical studies. In H2Eb-expressing transgenic mice, we identified three T cell epitopes from mouse Tg, mTg179, mTg409 and mTg2342, based on homology to epitopes hTg179, hTg410 and hTg2344, respectively, which we and others have found stimulatory or pathogenic in both DR3- and H2E-expressing mice. The high homology among these peptides with shared presentation by DR3, H2Eb and H2Ek molecules led us to examine the binding pocket residues of these class II molecules. Their similar binding characteristics help explain the pathogenic capacity of these T cell epitopes. Our approach of using appropriate human and murine MHC class II transgenic mice, combined with the synthesis and testing of potential pathogenic Tg peptides predicted from computational models of MHC-binding motifs, should continue to provide insights into human autoimmune thyroid disease. [Copyright &y& Elsevier]

Patients with previous percutaneous coronary intervention (PCI) are often excluded from clinical trials. As a result, limited data are available on the long-term outcome of such patients undergoing repeat PCI. In this study, we assessed the impact of previous PCI on outcomes in patients undergoing repeat PCI. We compared the baseline features and outcomes of 7,056 patients without previous PCI (group I) with those of 1,281 patients with previous PCI of the original target lesion (group II) and 1,408 patients with previous PCI of a nontarget lesion (group III) undergoing PCI in the Prevention of Restenosis with Tranilast and its Outcomes (PRESTO) trial. Compared with patients in group I, patients in groups II and III were more likely to have diabetes (25% and 24% vs 21%, p &#60;0.02), previous myocardial infarction (51% and 56% vs 29%, p &#60;0.001), and ostial lesions (10% and 7% vs 5%, p &#60;0.001), and less likely to have, as their indication for PCI, myocardial infarction (2% and 7% vs 17%, p &#60;0.001). At 1 month, major adverse cardiac events, including death, myocardial infarction, and repeat revascularization, were low and similar in all 3 groups. Compared with patients in group I, the risk of major adverse cardiac events at 9 months was significantly increased for patients in groups II (34.1% vs 18.6%, relative risk [RR] 2.03, adjusted RR 1.78, 95% confidence interval 1.58 to 2.01) and III (23.9% vs 18.6%, RR 1.30, adjusted RR 1.16, 95% confidence interval 1.02 to 1.33). The increased risk of major adverse cardiac events was entirely due to higher rates of repeat revascularization. In conclusion, despite similar short-term outcomes, patients with previous PCI undergoing PCI of either target or nontarget lesions had lower event-free survival at 9 months of follow-up. [Copyright &y& Elsevier]

Molecular imaging is an important new direction in medical diagnosis; however, its success is dependent upon molecular probes that demonstrate selective tissue targeting. We report the design and chemical synthesis of a derivative of human amyloid-β (Aβ) peptide that is capable of selectively targeting individual amyloid plaques in the brain of Alzheimer' s disease transgenic mice after being intravenously injected. This derivative is based on the sequence of the first 30 amino acid residues of Aβ with asparagyl/glutamyl-4-aminobutane residues (N-4ab/Q-4ab) substituted at unique Asp and Glu positions and with Gd-DTPA-aminohexanoic acid covalently attached at the N-terminal Asp. The Gd[N-4ab/Q-4ab]Aβ30 peptide was homogeneous as shown by high-resolution analytical techniques with a mass of ±4385 Da determined by electrospray ionization mass spectrometry. This diamine-and gadolinium-substituted derivative of Aβ is shown to have enhanced in vitro binding to Alzheimer' s disease (AD) amyloid plaques and increased in vivo permeability at the blood-brain barrier because of the unique Asp/Glu substitutions. In addition, specific in vivo targeting to AD amyloid plaques is demonstrated throughout the brain of an APP, PSl transgenic mouse after intravenous injection. Because of the magnetic resonance (MR) imaging contrast enhancement provided by gadolinium, this derivative should enable the in vivo MR imaging of individual amyloid plaques in the brains of AD animals or patients to allow for early diagnosis and also provide a direct measure of the efficacy of anti-amyloid therapies currently being developed. [ABSTRACT FROM AUTHOR]

Peptide nucleic acids (PNAs) are uncharged DNA analogs that hybridize to complementary sequences with high affinity and stability. We previously showed that PNAs, after intraperitoneal injection into rats, are effective antisense compounds in vivo. The present study was designed to test whether PNAs also have antigene effects in vivo. The renin-angiotensin system is critical in the control of blood pressure. We designed and synthesized sense (antigene) PNAs to angiotensinogen, which is the precursor protein that leads to angiotensin I and II. Spontaneously hypertensive rats received intraperitoneal injections of either 20 mg/kg sense-angiotensinogen-PNA, mismatch-angiotensinogen PNA, or saline. Only the sense-angiotensinogen PNA treatment resulted in a significant decrease in plasma angiotensin I, systolic blood pressure, and liver and brain angiotensinogen mRNA levels. Thus, these results demonstrate on the molecular, protein, and physiological levels that antigene PNAs are effective in vivo upon systemic administration. [Copyright &y& Elsevier]

Experimental autoimmune thyroiditis (EAT) can be induced in genetically susceptible mice by immunization with the self antigen, thyroglobulin (Tg). Since susceptibility is linked to H2 class II molecules, we have generated human leukocyte antigen (HLA) class II transgenic mice to study potential HLA associations with Hashimoto''s thyroiditis. DR3 (HLA-DRA/DRB1*0301) and DQ8 (HLA-DQA1*0301/DQB1*0302) transgenes were introduced into class II-negative Ab0/B10 and Ab0 nonobese diabetic (Ab0/NOD) mice. Previous work had shown that DR3 transgenic mice were susceptible to both mouse Tg and human Tg-induced EAT, whereas DQ8 transgenic mice were moderately susceptible only to human Tg induction. In this report, we examined the effect of DQ8 transgene on mouse Tg- and human Tg-induced EAT in double transgenic DR3/DQ8 mice. After mouse Tg induction, thyroiditis in DR3+DQ8+ Ab0/B10 mice was significantly less severe than in DR3+ mice but more severe than in DQ8+ mice. No difference in thyroiditis was observed between DR3+ and DR3+DQ8+ mice in another background strain, Ab0/NOD. However, after immunization with human Tg, DQ8 coexpression downregulated thyroiditis severity, compared to DR3+ mice, whereas thyroiditis was more extensive than in DQ8+ mice. Thus, depending on the background strain and the Tg used to induce disease, the presence of the DQ8 transgene can reduce thyroiditis mediated by DR3 molecules. [Copyright &y& Elsevier]

Glucagon metabolism in humans is poorly understood and is currently under active investigation. We have recently developed a novel isotope dilution method using nonradioactive, stable human glucagon (Phe 6 13C9, 15N; Phe 22 13C9, 15N) tracer to measure glucagon turnover. In this study we combined this method with organ catheterization technique to estimate splanchnic and leg glucagon extraction and uptake. We present data from the first 6 healthy subjects (age 25±3.6 yrs; BMI 26.7±4.5 kg/m2; fasting glucose 4.6±0.5 mM; HbA1c 5.1±0.3%) completed thus far. After IRB approval and informed consent, subjects were admitted in the morning after an overnight fast to Interventional Radiology, where catheters were placed in the right femoral artery and vein under local anesthesia with aseptic precautions. Under fluoroscopic guidance, a hepatic venous catheter was positioned through the femoral vein. Glucagon tracer was infused intravenously and Indocyanine Green infused via the femoral artery sheath to measure splanchnic and femoral plasma flows. Plasma samples were collected periodically from femoral artery, femoral vein and hepatic vein for concentrations of glucagon tracer (Tandem Mass Spectrometry), glucagon, glucose and Indocyanine Green (Spectrophotometry) concentrations. Fractional splanchnic glucagon extraction was 24.0±12.5 % while total splanchnic glucagon uptake was 214.3±56.1 ng/kg total body weight/min. Net splanchnic glucagon balance was -34.4±10.0 ng/kg total body weight/min implying net splanchnic glucagon production. Fractional leg glucagon extraction was 13.5±3.8% while total leg glucagon uptake was 53.0±21.2 pg/kg total body weight/min. To the best of our knowledge, these initial data provide the first direct assessment of regional glucagon metabolism in healthy humans in the post absorptive fasting state. Disclosure: F. Ruchi: None. Y.R. Yadav: None. D. Romeres: None. S. Sawleh: None. L.M. Benson: None. K.L. Johnson: None. C. Dalla Man: Research Support; Self; Sanofi-Aventis Deutschland GmbH. C. Cobelli: None. D.J. McCormick: None. L.R. Wilkins: None. R. Basu: Consultant; Self; GENFIT. Research Support; Self; AstraZeneca. A. Basu: Consultant; Spouse/Partner; GENFIT. Research Support; Spouse/Partner; AstraZeneca. Funding: National Institutes of Health (R01DK029953 to R.B.), (R01DK085516 to A.B.), (DK059637, DK020593) [ABSTRACT FROM AUTHOR]

Glucagon metabolism in humans is poorly understood and is currently under active investigation. We have recently developed a new isotope dilution method using nonradioactive, stable human glucagon (Phe 6 13C9, 15N; Phe 22 13C9, 15N) tracer to measure glucagon fluxes. To estimate postprandial glucagon turnover using this novel technique, we present data from the first 5 healthy subjects (3 males, age 26.2±7.9 years, BMI 25.4±2.6 kg/m2, fasting plasma glucose 4.7±0.2 mM, HbA1c 5.1±0.2 %) done thus far. After IRB approval and informed consent, subjects presented to the clinical research unit in the morning after an overnight fast. A catheter was inserted in a forearm vein for glucagon tracer infusion and another catheter placed in the contralateral hand and the hand kept in a heated box for periodic draws of arterialized venous samples for measurements of glucagon tracer (tandem mass spectrometry), glucagon and glucose concentrations during the study. A mixed meal (75 grams carb, 15% protein, 35% fat; 8 kcal/kg) was ingested at time 0 and the glucagon tracer infusion rate adjusted to mimic the anticipated changes in postprandial circulating glucagon concentrations for 6 hours. Systemic glucagon appearance rate (Ra glucagon) was calculated by the isotope dilution method. Fig 1 shows the Ra glucagon in all subjects. These initial data show that postprandial glucagon turnover can be calculated in humans applying this novel technique. Disclosure: F. Ruchi: None. Y.R. Yadav: None. D. Romeres: None. S. Sawleh: None. L.M. Benson: None. K.L. Johnson: None. M. Schiavon: None. C. Dalla Man: Research Support; Self; Sanofi-Aventis Deutschland GmbH. C. Cobelli: None. D.J. McCormick: None. R. Basu: Consultant; Self; GENFIT. Research Support; Self; AstraZeneca. A. Basu: Consultant; Spouse/Partner; GENFIT. Research Support; Spouse/Partner; AstraZeneca. Funding: National Institutes of Health (R01DK029953 to R.B.), (R01DK085516 to A.B.), (DK059637, DK020593) [ABSTRACT FROM AUTHOR]

We have recently developed a novel isotope dilution method using nonradioactive, stable human Glucagon (Phe 6 13C9, 15N; Phe 22 13C9, 15N) tracer to measure glucagon turnover. In this study we combined this method with organ catheterization technique to estimate the effects of increasing circulating glucagon concentrations, spanning the physiological range, on splanchnic and leg glucagon extraction. We present data from the first 6 healthy subjects (age 25±3.6 yrs; BMI 26.7±4.5 kg/m2; fasting glucose 4.6±0.5 mM; HbA1c 5.1±0.3%) completed thus far. After IRB approval and informed consent, subjects were admitted in the morning after an overnight fast to Interventional Radiology, where catheters were placed in the right femoral artery and vein under local anesthesia with aseptic precautions. Under fluoroscopic guidance, a hepatic venous catheter was positioned through the femoral vein. Glucagon tracer was infused intravenously and Indocyanine Green infused via the femoral artery sheath to measure splanchnic and femoral plasma flows. After a 90 min period for basal sampling, exogenous glucagon was infused at 0.65 ng/kg/min for 60 minutes (low) followed by 1.5 ng/kg/min (high) for another 60 minutes. Plasma samples were collected periodically from femoral artery, femoral vein and hepatic vein for concentrations of Glucagon tracer (Tandem Mass Spectrometry), glucagon, glucose and Indocyanine Green (Spectrophotometry) concentrations. Arterial glucagon concentrations were 45.2±9.6 pg/ml (basal), 56.3±13.3 pg/ml (low) and 81.4±15.5 pg/ml (high). While fractional splanchnic glucagon extraction remained relatively unchanged (24.0±12.5% vs. 27.7±15.6% vs. 23.5±10.1%: basal vs. low vs. high), fractional leg glucagon extraction tended to increase (13.5±3.8% vs. 16.0±7.2% vs. 18.5±9.4%: basal vs. low vs. high) with increasing glucagon concentrations. These initial data suggest differing mechanisms of glucagon clearance across the splanchnic and leg tissues in humans. Disclosure: F. Ruchi: None. Y.R. Yadav: None. D. Romeres: None. S. Sawleh: None. L.M. Benson: None. K.L. Johnson: None. C. Dalla Man: Research Support; Self; Sanofi-Aventis Deutschland GmbH. C. Cobelli: None. D.J. McCormick: None. L.R. Wilkins: None. R. Basu: Consultant; Self; GENFIT. Research Support; Self; AstraZeneca. A. Basu: Consultant; Spouse/Partner; GENFIT. Research Support; Spouse/Partner; AstraZeneca. Funding: National Institutes of Health (R01DK029953 to R.B.), (R01DK085516 to A.B.), (DK059637, DK020593) [ABSTRACT FROM AUTHOR]

Background: Glucagon serves as an important regulatory hormone for regulating blood glucose concentration with tight feedback control exerted by insulin and glucose. There are critical gaps in our understanding of glucagon kinetics, pancreatic α cell function and intra-islet feedback network that are disrupted in type 1 diabetes. This is important for translational research applications of evolving dual-hormone (insulin + glucagon) closed-loop artificial pancreas algorithms and their usage in type 1 diabetes. Thus, it is important to accurately measure glucagon kinetics in vivo and to develop robust models of glucose-insulin-glucagon interplay that could inform next generation of artificial pancreas algorithms., Methods: Here, we describe the administration of novel 13 C 15 N heavy isotope-containing glucagon tracers-FF glucagon [(Phe 6 13 C 9 , 15 N; Phe 22 13 C 9 , 15 N)] and FFLA glucagon [(Phe 6 13 C 9 , 15 N; Phe 22 13 C 9 , 15 N; Leu 14 13 C 6 , 15 N; Ala 19 13 C 3 )] followed by anti-glucagon antibody-based enrichment and LC-MS/MS based-targeted assays using high-resolution mass spectrometry to determine levels of infused glucagon in plasma samples. The optimized assay results were applied for measurement of glucagon turnover in subjects with and without type 1 diabetes infused with isotopically labeled glucagon tracers., Results: The limit of quantitation was found to be 1.56 pg/ml using stable isotope-labeled glucagon as an internal standard. Intra and inter-assay variability was < 6% and < 16%, respectively, for FF glucagon while it was < 5% and < 23%, respectively, for FFLA glucagon. Further, we carried out a novel isotope dilution technique using glucagon tracers for studying glucagon kinetics in type 1 diabetes., Conclusions: The methods described in this study for simultaneous detection and quantitation of glucagon tracers have clinical utility for investigating glucagon kinetics in vivo in humans., (© 2022. The Author(s).)

Multiple Myeloma (MM) patients suffer disease relapse due to the development of therapeutic resistance. Increasing evidence suggests that immunotherapeutic strategies can provide durable responses. Here we evaluate the possibility of adoptive cell transfer (ACT) by generating ex vivo T cells from peripheral blood mononuclear cells (PBMCs) isolated from MM patients by employing our previously devised protocols. We designed peptides from antigens (Ags) including cancer testis antigens (CTAs) that are over expressed in MM. We exposed PBMCs from different healthy donors (HDs) to single peptides. We observed reproducible Ag-specific cluster of differentiation 4 + (CD4 + ) and CD8 + T cell responses on exposure of PBMCs to different single peptide sequences. These peptide sequences were used to compile four different peptide cocktails. Naïve T cells from PBMCs from MM patients or HDs recognized the cognate Ag in all four peptide cocktails, leading to generation of multiclonal Ag-specific CD4 + and CD8 + effector and central memory T (T EM and T CM , respectively) cells which produced interferon-gamma (IFN-γ), granzyme B and perforin on secondary restimulation. Furthermore, this study demonstrated that immune cells from MM patients are capable of switching metabolic programs to induce effector and memory responses. Multiple peptides and cocktails were identified that induce IFN-γ + , T1-type, metabolically active T cells, thereby paving the way for feasibility testing of ACT in phase I clinical trials., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest., (Copyright: © 2021 Vardam-Kaur et al.)

A key immunomodulatory cytokine, IL-10 (interleukin-10), has been shown to be dysregulated in preeclampsia, a pregnancy-specific hypertensive disorder, further characterized by multi-system involvement. However, studies have reported inconsistent findings about circulating IL-10 levels in preeclamptic versus normotensive pregnancies. The aim of the present systematic review and meta-analysis was to assess circulating IL-10 levels in preeclamptic and normotensive pregnancies at 2 time points: before, and at the time of preeclampsia diagnosis. PubMED, EMBASE, and Web of Science databases were searched to include all published studies examining circulating IL-10 levels in preeclamptic and normotensive pregnancies. Differences in IL-10 levels were evaluated by standardized mean differences. Of 876 abstracts screened, 56 studies were included in the meta-analysis. Circulating IL-10 levels were not different before the time of active disease (standardized mean differences, -0.01 [95% CI, -0.11 to 0.08]; P =0.76). At the time of active disease, women with preeclampsia (n=1599) had significantly lower IL-10 levels compared with normotensive controls (n=1998; standardized mean differences, -0.79 [95% CI, -1.22 to -0.35]; P =0.0004). IL-10 levels were lower in both early/severe and late/mild forms of preeclampsia. Subgroup analysis revealed that IL-10 measurement methodology (ELISA or multiplex bead array) and the sample type (plasma or serum) significantly influenced the observed differences, with the use of sera paired with ELISA technology providing the best distinction in IL-10 levels between preeclamptic and normotensive pregnancies. These findings support the role of decreased IL-10 levels in the pathophysiology of preeclampsia. Future studies should address the therapeutic potential of IL-10 in preeclampsia.

Macropinocytosis supports the metabolic requirement of RAS-transformed pancreatic ductal adenocarcinoma cells (PDACs). However, regulators of RAS-transformation (activation) that lead to macropinocytosis have not been identified. Herein, we report that UBAP2 (ubiquitin-binding associated protein 2), regulates the activation of KRAS and macropinocytosis in pancreatic cancer. We demonstrate that UBAP2 is highly expressed in both pancreatic cancer cell lines and tumor tissues of PDAC patients. The expression of UBAP2 is associated with poor overall survival in several cancers, including PDAC. Silencing UBAP2 decreases the levels of activated KRAS, and inhibits macropinocytosis, and tumor growth in vivo. Using a UBAP2-deletion construct, we demonstrate that the UBA-domain of UBAP2 is critical for the regulation of macropinocytosis and maintaining the levels of activated KRAS. In addition, UBAP2 regulates RAS downstream signaling and helps maintain RAS in the GTP-bound form. However, the exact mechanism by which UBAP2 regulates KRAS activation is unknown and needs further investigation. Thus, UBAP2 may be exploited as a potential therapeutic target to inhibit macropinocytosis and tumor growth in activated KRAS-driven cancers., (© 2020 Federation of American Societies for Experimental Biology.)

The polycystin-1 (PC1), polycystin-2 (PC2) and fibrocystin proteins, the respective products of the PKD1, PKD2 and PKHD1 genes, are abundant in urinary exosome-like vesicles (ELVs) where they form the polycystin complex (PCC). ELVs are 100 nm diameter membrane vesicles shed into the urine by the cells lining the nephron. Using MS/MS analysis of ELVs from individuals with PKD1 mutations and controls, we show that in addition to the well-described GPS/GAIN cleavage event in PC1 at 3048 aa and the proprotein convertase cleavage (PPC) event in fibrocystin at 3616 aa, there are multiple other cleavage events in these proteins. The C-terminal 11 transmembrane portion of PC1 undergoes three cleavage events in vivo. The absence of peptides from the C-terminal cytoplasmic tail of fibrocystin implies a cleavage event close to its single TM domain prior to loading onto the ELVs. There is also evidence that the C-terminal tail of PC2 is also cleaved in ELVs. Native gel analysis of the PCC shows that the entire complex is  > 2 MDa in size and that N-terminal GPS/GAIN cleaved PC1 and PPC cleaved fibrocystin ectodomains can be released under non-reducing conditions and resolve at 300 kDa. This paper shows that the three major human cystogene proteins are detectable in human urinary ELVs and that all three undergo post-translational proteolytic processing. Human urinary ELVs may be a useful source of material in the search for proteins that interact with the PCC.

Despite optimal current therapies, cardiovascular disease remains the leading cause for death worldwide. Importantly, advances in peptide engineering have accelerated the development of innovative therapeutics for diverse human disease states. Additionally, the advancement of bispecific therapeutics targeting >1 signaling pathway represents a highly innovative strategy for the treatment of cardiovascular disease. We, therefore, engineered a novel, designer peptide, which simultaneously targets the pGC-A (particulate guanylyl cyclase A) receptor and the MasR (Mas receptor), potentially representing an attractive cardiorenoprotective therapeutic for cardiovascular disease. We engineered a novel, bispecific receptor activator, NPA7, that represents the fusion of a 22-amino acid sequence of BNP (B-type natriuretic peptide; an endogenous ligand of pGC-A) with Ang 1-7 (angiotensin 1-7)-the 7-amino acid endogenous activator of MasR. We assessed NPA7's dual receptor activating actions in vitro (second messenger production and receptor interaction). Further, we performed an intravenous peptide infusion comparison study in normal canines to study its biological actions in vivo, including in the presence of an MasR antagonist. Our in vivo and in vitro studies demonstrate the successful synthesis of NPA7 as a bispecific receptor activator targeting pGC-A and MasR. In normal canines, NPA7 possesses enhanced natriuretic, diuretic, systemic, and renal vasorelaxing and cardiac unloading properties. Importantly, NPA7's actions are superior to that of the individual native pGC-A or MasR ligands. These studies advance NPA7 as a novel, bispecific designer peptide with potential cardiorenal therapeutic benefit for the treatment of cardiovascular disease, such as hypertension and heart failure.

Coronary artery bypass grafting (CABG) decreases mortality in patients with significant left main (LM) coronary artery disease and for years remained the therapy of choice for patients with this ominous lesion. Advances in percutaneous coronary intervention (PCI) have enabled it to become an alternative to CABG. The results of observational registries and randomized comparisons have shown the safety and efficacy of PCI in appropriately selected patients with low or intermediate angiographic risk scores. Furthermore, the use of physiological measures of flow limitation and the use of intracoronary imaging techniques has added benefit and improved outcomes. The use of fractional flow reserve to more accurately evaluate the significance of intermediate lesions and guide the extent of revascularization has been an important refinement. Intravascular ultrasound and optical coherence tomography assessment of optimal stent deployment has led to reductions in restenosis. Newer generation stents, combined with improvements in specific techniques, especially at the LM bifurcation have extended PCI to more complex anatomic scenarios. The availability of left ventricular support devices in patients with complex coronary anatomy and severely depressed left ventricular function has added a margin of safety to LM and multivessel intervention. Randomized comparisons of CABG with PCI in carefully selected patients, using contemporaneous surgical and interventional techniques and optimal medical therapy, will further aid heart teams in the decision-making process. In conclusion, this review will give a concise overview of the management of unprotected LM disease., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objectives: The aim of this study was to compare trends and outcomes of 3 approaches to carotid revascularization in the coronary artery bypass graft (CABG) population when performed during the same hospitalization., Background: The optimal approach to managing coexisting severe carotid and coronary disease remains controversial. Carotid endarterectomy (CEA) or carotid artery stenting (CAS) are used to decrease the risk of stroke in patients with carotid disease undergoing CABG surgery., Methods: The authors conducted a serial, cross-sectional study with time trends of 3 revascularization groups during the same hospital admission: 1) combined CEA+CABG; 2) staged CEA+CABG; and 3) staged CAS+CABG from the Nationwide Inpatient Sample database 2004 to 2012. The primary composite endpoints were in-hospital all-cause death, stroke, and death/stroke., Results: During the 9-year period, 22,501 concurrent carotid revascularizations and CABG surgeries during the same hospitalization were performed. Of these, 15,402 (68.4%) underwent combined CEA+CABG, 6,297 (28.0%) underwent staged CEA+CABG, and 802 (3.6%) underwent staged CAS+CABG. The overall rate of CEA+CABG decreased by 16.1% (p trend  = 0.03) from 2004 to 2012, whereas the rate of CAS+CABG did not significantly change during these years (p trend  = 0.10). The adjusted risk of death was greater, whereas risk of stroke was lower with both combined CEA+CABG (death odds ratio [OR]: 2.08, 95% confidence interval [CI]: 1.08 to 3.97; p = 0.03; stroke OR: 0.65, 95% CI: 0.42 to 1.01; p = 0.06) and staged CEA+CABG (death OR: 2.40, 95% CI: 1.43 to 4.05; p = 0.001; stroke OR: 0.50, 95% CI: 0.31 to 0.80; p = 0.004) approaches compared with CAS+CABG. The adjusted risk of death or stroke was similar in the 3 groups., Conclusions: In patients with concomitant carotid and coronary disease undergoing combined revascularization, combined CEA+CABG is utilized most frequently, followed by staged CEA+CABG and staged CAS+CABG strategies. The staged CAS+CABG strategy was associated with lower risk of mortality, but higher risk of stroke. Future studies are needed to examine the risks/benefits of different carotid revascularization strategies for high-risk patients requiring concurrent CABG., (Copyright © 2017 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Aims: Cenderitide is a novel dual natriuretic peptide (NP) receptor chimeric peptide activator, which targets the particulate guanylyl cyclase B (pGC-B) receptor and pGC-A unlike native NPs. Cenderitide was engineered to retain the anti-fibrotic properties of C-type natriuretic peptide (CNP)/pGC-B with renal-enhancing actions facilitated by fusion to the carboxyl terminus of Dendroaspis NP (DNP), a pGC-A agonist, to CNP. Here, we address significance of the DNP carboxyl terminus in dual pGC receptor activation and actions of cenderitide compared with CNP on renal function and cyclic guanosine monophosphate (cGMP) in vivo and ex vivo in normal canines., Methods and Results: In vitro, only cenderitide and not CNP or three CNP-based variants was a potent dual pGC-A/pGC-B activator of cGMP production (from 5 to 237 pmol/mL) in human embryonic kidney (HEK) 293 cells overexpressing human pGC-A while in pGC-B overexpressing cells cenderitide increased cGMP production (from 4 to 321 pmol/mL) while the three CNP-based variants were weak agonists. Based upon our finding that the DNP carboxyl terminus is a key structural requirement for dual pGC-A/pGC-B activation, we defined in vivo the renal-enhancing actions of cenderitide compared with CNP. Cenderitide increased urinary cGMP excretion (from 989 to 5977 pmol/mL), net generation of renal cGMP (821-4124 pmol/min), natriuresis (12-242 μEq/min), and glomerular filtration rate (GFR) (37-51 mL/min) while CNP did not. We then demonstrated the transformation of CNP ex vivo into a renal cGMP-activating peptide which increased cGMP in freshly isolated glomeruli eight-fold greater than CNP., Conclusion: The current study establishes that dual pGC-A and pGC-B activation with CNP requires the specific carboxyl terminus of DNP. In normal canines in vivo and in glomeruli ex vivo, the carboxyl terminus of DNP transforms CNP into a natriuretic and GFR-enhancing peptide. Future studies of cenderitide are warranted in cardiorenal disease states to explore its efficacy in overall cardiorenal homeostasis., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.)

While coronary artery bypass grafting (CABG) has been the standard of care for patients with unprotected left main coronary artery disease, advances in percutaneous coronary intervention (PCI) have made stent placement a reasonable alternative in selected patients. In this review, we address the results of studies comparing PCI with CABG, discuss the invasive evaluation of these patients, and the technical approach to percutaneous revascularization. Furthermore, we discuss future pivotal trials, which will help define long-term outcomes comparing PCI with surgery.

As percutaneous coronary intervention (PCI) is being applied to higher-risk patients, ie, those with unprotected left main, multi-vessel disease, last remaining vessel, compromised left ventricular function, and ongoing ischemia, interventional cardiologists have used different percutaneous assist devices in an attempt to reduce procedure risk. The definition of high risk has varied among trials. There is no definitive evidence for superiority of the more invasive devices over the intra-aortic balloon pump (IABP); furthermore, a prophylactic strategy of IABP insertion has not proven superior to a provisional strategy. The purpose of this report is to review the physiologic mechanism of action of the devices and discuss indications, limitations, and clinical outcomes during high-risk PCI.

Objective: The multicenter EPIC (FiberNet Embolic Protection System in Carotid Artery Stenting Trial) single-arm trial evaluated the 30-day outcomes of a new design concept for embolic protection during carotid artery stenting (CAS)., Background: Embolic protection filters available for use during CAS include fixed and over-the-wire systems that rely on embolic material capture within a "basket" structure. The FiberNet Embolic Protection System (EPS), which features a very low crossing profile, consists of a three-dimensional fiber-based filter distally mounted on a 0.014 inch guidewire with integrated aspiration during filter retrieval., Methods: The trial enrolled 237 patients from 26 centers. Demographics, clinical and lesion characteristics, as well as adverse events through a 30-day follow-up were recorded. The mean age of the patients was 74 years, 64% were male and 20% had symptomatic carotid artery disease., Results: The combined major adverse event (MAE) rate at 30 days for all death, stroke, and myocardial infarction was 3.0%. There were three major strokes (two ischemic and one hemorrhagic) and two minor strokes (both ischemic) for a 2.1% 30-day stroke rate. The procedural technical success rate was 97.5% and macroscopic evidence of debris was reported in 90.9% of the procedures., Conclusions: The FiberNet EPS, used with commercially available stents, produced low stroke rates following CAS in high surgical risk patients presenting with carotid artery disease. The unique filter design including aspiration during retrieval may have contributed to the low 30-day stroke rate reported during CAS in patients considered at high risk for complications following carotid endarterectomy (CEA)., (Copyright 2010 Wiley-Liss, Inc.)

Stroke is the third leading cause of death in the United States. Carotid artery stenosis represents one of the most common etiologies of stroke. The current treatment modalities available for the treatment of carotid artery stenosis are carotid endarterectomy (CEA) and carotid artery stenting (CAS). Several clinical trials comparing CEA with medical management showed superiority of the surgical arm; however, the applicability of these results to the general population is limited by the fact that the patients and surgeons enrolled in these trials were carefully selected, and the optimal medical therapy used does not meet the current treatment standards. Carotid artery stenting has emerged as a treatment alternative to CEA, as shown in randomized trials comparing the 2 treatment modalities. Recent data from large-volume CAS registries indicate that percutaneous treatment of carotid artery stenosis compares favorably to CEA. Furthermore, the CAS trial designs make these results more applicable to the community standards. These data suggest that CAS will become the treatment of choice in patients with carotid artery stenosis.

Mantle-cell lymphoma (MCL) is a unique subtype of B-cell non-Hodgkin lymphoma (NHL) that behaves aggressively and remains incurable. In order to understand the pathogenesis of MCL and design new therapies, it is important to accurately analyze molecular changes in pathways dysregulated in MCL. We used antibody microarrays to compare patterns of protein expression between CD19(+) purified B lymphocytes from normal tonsil and 7 cases of histologically confirmed MCL. Protein overexpression was defined as a higher than 1.3-fold or 2-fold increase in at least 67% of tumor samples compared with normal B-cell control. Of the polypeptides, 77 were overexpressed using the higher than 1.3-fold cutoff, and 13 were overexpressed using the 2-fold cutoff. These included cell cycle regulators (regulator of chromosome condensation 1 [RCC1], murine double minute 2 [MDM2]), a kinase (citron Rho-interacting kinase [CRIK]), chaperone proteins (heat shock 90-kDa protein [Hsp90], Hsp10), and phosphatase regulators (A-kinase anchor protein 1 [AKAP149], protein phosphatase 5 [PP5], and inhibitor 2). The elevated expression of some of these polypeptides was confirmed by immunoblotting and immunohistochemistry, whereas elevated expression of others could not be confirmed, illustrating the importance of confirmatory studies. This study describes a novel technique that identifies proteins dysregulated in MCL.

A novel chemical-enzymatic approach was developed to facilitate identification of phosphorylation sites in isolated phosphoproteins. ESI-TOF mass spectrometry was used to characterize products from the chemical-enzymatic cleavage of specific phosphorylation sites in bovine alpha-S1 casein and synthetic phosphopeptides containing substitutions at a single phosphorylation site. Further refinements to this approach for identification of protein phosphorylation sites and its utility for the quantification of phosphopeptides by isotope-dilution mass spectrometry are presented.

Synovial fluid (SF) is a dynamic reservoir for proteins originating from serum, synovial tissue, and cartilage. The composition of the SF proteome may reflect the pathophysiological conditions affecting the circulatory system and cartilage. Our long-term goal is to identify reliable protein markers for osteoarthritis (OA) in SF. We first evaluated the pattern of SF proteins on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) as a function of protein loading, pH range for isoelectric focusing, and concentration of acrylamide in SDS-PAGE. Removal of albumin and Gamma-globulins from the samples did not improve the detection of protein spots on 2D-PAGE. The repeatability of protein spot intensity was tested by triplicate 2D-PAGE of a given sample; these experiments showed low intrasample variability (correlation coefficients 0.89-0.95). Differences between multiple samples were tested by comparing the 2D-PAGE of four samples. These experiments showed slightly greater variation between samples (correlation coefficients 0.85-0.93) and a number of differentially expressed proteins. The intensity of 18 protein spots differed more than fivefold, and the intensity of nine protein spots differed more than 100-fold. These results show that 2D-PAGE can be used under standard conditions to screen SF samples and identify a small subset of proteins in SF that are potential markers associated with OA.